The Flaviviridae are a family of positive-sense RNA viruses that contain numerous important human pathogens. Many of the molecular mechanisms that underlie how these RNA viruses cause cytopathology and disease are not clearly described. The cellular mRNA decay machinery, in particularly the 5'-3' pathway mediated by the exoribonuclease Xrn1, plays a major role in regulating the abundance and quality of gene expression in the cell. Understudied, but nevertheless very important aspects of flavivirus-host interactions, include how viral RNAs are protected from degradation by the cellular mRNA decay machinery and what are the implications of the viral RNA stabilization strategies on the regulation of cellular mRNA stability. We have recently observed that flaviviruses repress the activity of Xrn1 through trapping the enzyme using unique structured regions of the viral RNA. Interestingly, it is the 5' UTR IRES region that is responsible for this Xrn1 repression in Hepatitis C virus and Bovine Viral Diarrhea virus. These observations serve as the foundation for this proposal to gain in-depth mechanistic insights into molecular mechanisms of Xrn1 repression and regulation that are disrupted by flavivirus RNAs. In Aim 1, we will identify the sequence/structural requirements of IRES-mediated Xrn1 repression and determine whether this is a common property of other viral IRES elements. The goal of Aim 2 is understand at a mechanistic level why the repression of Xrn1 by flavivirus RNAs results in the apparent shut down of the entire 5'-3' mRNA decay pathway ? not just the exonucleolytic digestion step. Uncovering the interplay and feedback regulation of the decay factors in the 5'-3' RNA decay pathway will provide novel insights into how the cell normally regulates this decay pathway and integrate it into the overall process of gene expression. In the third aim we will expand our studies on Xrn1 stalling and investigate whether it is an approach used by the cell to remodel cellular transcripts. In the final Aim, we will characterize key biological aspects of Xrn1 repression from the perspective of both the cell and the virus. A key focus of this part will be on the dysregulation of cellular mRNA stability by flaviviruses that results in dramatic changes in cellular gene expression that could play a significant role in HCV-mediated oncogenesis.